Protein-polycation nucleic acid complexes and methods of use

ABSTRACT

The invention relates to a system for transporting nucleic acids into the cell, which is effected by receptor-mediated endocytosis. Using a transferrin-polycation conjugate, a complex can be formed with the polyanionic nucleic acid. This complex is bound to the transferrin receptor, which is highly regulated in growing cells, and absorbed into the cell. Suitable nucleic acids include those which inhibit specific genes or the RNA function, such as antisense oligonucleotides or ribozymes or the genes coding for them. The invention further relates to a process for introducing nucleic acids into the cells, transferrin-polycation/nucleic acid complexes and pharmaceutical preparations containing them.

This application is a division, of application Ser. No. 07/492,460,filed Mar. 9, 1990, now U.S. Pat. No. 5,354,.

FIELD OF THE INVENTION

The invention relates to the transporting of substances with an affinityfor polycations, particularly nucleic acids, into the cell, thetransportation being carried out by means of receptor-mediatedendocytosis.

BACKGROUND OF THE INVENTION

Antisense RNAs and DNAs have proved to be effective agents forselectively inhibiting certain genetic sequences in cell-free systems aswell as within the living cell. Their mode of activity is based on thespecific recognition of a complementary nucleic acid strand andattachment thereto, thus affecting the transcription, translation andcleaving processes. This mechanism of activity theoretically makes itpossible to use antisense oligonucleotides as therapeutic agents whichwill block the expression of certain genes (such as deregulatedoncogenes or viral genes) in vivo. It has already been shown that shortantisense oligonucleotides can be imported into cells and perform theirinhibiting activity therein (Zamecnik et al., 1986), even though theintracellular concentration thereof is low, partly because of theirrestricted uptake through the cell membrane owing to the strong negativecharge of the nucleic acids.

Another method of selectively inhibiting genes consists in theapplication of ribozymes, i.e. RNA molecules which recognise specificRNA sequences and are able to bind to them and cleave them. Here againthere is the need to guarantee the highest possible concentration ofactive ribozymes in the cell, for which transportation into the cell isone of the limiting factors.

In order to counteract this limiting factor, a number of solutions havealready been proposed.

One of these solutions consists in direct modification of nucleic acids,e.g. by substituting the charged phosphodiester groups with unchargedmethyl phosphonates (Smith et al., 1986), carbamates (Stirchak et al.,1987) or silyl compounds (Cormier et al., 1988) or usingphosphorothioates (Stern et al., 1988). Another possible method ofdirect modification consists in the use of nucleoside analogues (Morvanet al., 1988, Praseuth et al., 1988)).

Even though some of these proposals appear to offer a promising way ofsolving the problem, they do have numerous disadvantages, e.g. reducedbinding to the target molecule and a reduced inhibitory effect. A chiefdisadvantage of the in vivo use of modified oligonucleotides is thepossible toxicity of these compounds.

An alternative method to the direct modification of the oligonucleotidesconsists in leaving the oligonucleotide itself unchanged and providingit with a group which will impart the desired properties to it, e.g.with molecules which will make transportation into the cell easier. Oneof the proposed solutions within the scope of this principle consists inconjugating the oligonucleotide with polycationic compounds (Lemaitre etal., 1987).

Various techniques are known for genetic transformation of mammaliancells in vitro, but the use of these techniques in vivo is restricted(these include the introduction of DNA by means of viruses, liposomes,electroporation, microinjection, cell fusion, DEAE dextran or thecalcium phosphate precipitation method).

Attempts have therefore already been made to develop a soluble systemwhich can be used in vivo, which will convey DNA into the cells in adirected manner by means of receptor-mediated endocytosis (G. Y. Wu, C.H. Wu, 1987). This system was developed for hepatocytes and is basedessentially on the following two facts:

1. On the surface of the hepatocytes there are receptors which bindspecific glycoproteins and convey them into the cell.

2. DNA can be bound to polycationic compounds, e.g. polylysine, by astrong electrostatic interaction, forming soluble complexes.

The system is based on the principle of coupling polylysine with aglycoprotein of a kind to which the receptor will respond and then, byadding DNA, forming a soluble glycoprotein/polylysine/DNA complex whichwill be transported to the cells containing the glycoprotein receptorand, after the absorption of the complex into the cell, will make itpossible for the DNA sequence to be expressed.

SUMMARY OF THE INVENTION

The objective of the present invention was to provide a highly activetransporting system which is more widely applicable than the knownsystem, which can only be used for one particular cell type owing to thespecific presence of the receptor on this one cell type.

According to the invention, the solution to the problem consists in thefact that the polycation is bound to transferrin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Ion exchange gradient separation of non-conjugated transferrinfrom transferrin-polylysine conjugates.

FIG. 2A and B. SDS gel electrophoresis showing an approximately equalcontent of transferrin in transferrin 1-polylysine conjugate fractionsafter pre-treatment with 2-mercaptoethanol. 2B. In unreduced samplesthere were no visible bands for transferrin, only less widely migratingconjugates. (T=untreated transferrin; 1-6=conjugate fractions 1-6.)

FIG. 3 (panels (A) and (B)). Preparation of transferrin-protamineconjugates. The transferrin-protamine conjugate fractions A-D are moreslowly migrating bands on SDS gel electrophoresis (A), whereas inβ-mercaptoethanol-reduced samples only the transferrin band was visible(B).

FIG. 4. Binding of transferrin-polycation conjugates with DNA wasconfirmed using a gel mobility shift assay using ³² P-labelled lambdaDNA cut with Eco R1/Hind III.

FIG. 5 (panels A, B and C). Fluorescence images of chicken erythroblastsincubated for 24 hours without (A) or with (B, C) FITC-labelledtransferrin-polylysine conjugates.

FIG. 6 (panels A and B). Erythroid differentiation as a function of theconcentration of transferrin or transferrin-polylysine.

(A) Hemoglobin content of cells grown in the presence of no additives(triangles); in the presence of iron-saturated conalbumin (circles); andin the presence of iron-saturated TfpL 270 (squares). The shaded areashows the hemoglobin content of cells grown without transferrin.

(B) Hemoglobin content of cells grown in the presence of iron-saturatedconalbumin (open circles); TfpL 90 (open squares) and TfpL 270 (solidsquares).

FIG. 7. Structure of transported DNA in comparison with tDNA ribozymes.

FIG. 8. Absorption of plasmid-DNA into chicken erythroblasts usingpolylysine-transferrin conjugates. (Circles: Tfprot; Squares: TfpL.)

FIG. 9. Absorption of DNA into chicken erythroblasts usingpolylysine-transferrin conjugates. (Circles: Tfprot; Squares: TfpL.)

FIGS. 10A and 10B. Absorption of transferrin-polycation/DNA complexesinto cells is effected via transferrin receptors.

(A) Luciferase activity achieved for pL-DNA complexes and TfpL-DNAcomplexes.

(B) Free transferrin in medium competes for DNA uptake mediated by TfpL.

FIG. 11(A-C). Structure of tRNA ribozyme genes directed againsttranslation initiation region of erbB.

FIG. 12. Absorption of DNA by cells treated withtransferrin-polylysine/DNA:

Trace m: molecular weight marker: pBR322 DNA, cleaved with HpaIl andradioactively labelled using the Klenow fragment of DNA polymerase withalpha-³² P-CTP.

Trace 1: 2000 cpm ES13 fragment.

Trace 2: material from cells treated with transferrin and ES13.

Trace 3: material from cells treated with transferrin-polylysine andES13.

FIG. 13. Binding and internalization of transferrin-polylysine/DNAcomplexes in hemopoietic chicken cells. Labelled TfpL 90 (squares) orlabelled TfpL 90 complexed with DNA (triangles) bound to HD3 cells insuch a way that saturation occurs.

FIG. 14. Uptake of TfpL conjugate and TfpL-DNA complex by cells. Cellswere incubated with Tf ( . . . ); TfpL ( . . . ); TfpL/DNA (₋₋); bindingbuffer ( . . . ).

FIG. 15. Uptake of TfpL/DNA complexes (using pRSV-βGal plasmid DNA) byHD3 cells. Cells were incubated with TfpL-pB-SK-DNA ( . . . ) or withTfpL-pRSV-βGal-DNA (₋₋). The fluorescent βGal substrate FDG wasintroduced by osmotic shock.

DETAILED DESCRIPTION OF THE INVENTION

Transferrins are a class of related metal-binding transportingglycoproteins with an in vivo specificity for iron. Various mammaliantransferrins have been identified, plasma transferrin supplying themajority of body tissue with iron. The main producer of transferrin isthe liver.

Transferrin has a molecular weight of about 80,000, 6% of which consistsof the sugar residues. A transferrin molecule is able to bind twomolecules of iron, and this binding requires the presence of carbonateor bicarbonate ions.

The transferrin receptor, which occurs in a number of forms whichpossibly deviate slightly from one another on the various cells (e.g. inthe carbohydrate group) is a transmembrane glycoprotein with a molecularweight of about 180,000, whilst a receptor molecule is able to bind oneor possibly two molecules of transferrin.

At the physiological pH of 7.4, the transferrin receptor has a very highaffinity for Fe₂ transferrin, lower affinity for the Fe transferrin andvirtually no affinity for apotransferrin, even though the latter forms avery stable complex with the receptor at a pH of about 5.

Transferrin receptors have been detected in particularly large numbersin precursors of erythrocytes, placenta and liver and in measurableamounts in a number of other body tissues. A particularly interestingobservation is that the receptor is highly regulated in growing cells.It has also been observed that the number of receptors is substantiallyincreased in neoplastic tissue in vivo compared with benign lesions,indicating an increased iron requirement. The absorption mechanism ofthe transferrin-iron complex by receptors and the intracellular cyclethereof have been thoroughly researched.

There is still no absolute certainty as to the exact sequence of eventsof the release of the iron molecules by transferrin, although themajority view is that the molecules are released mainly intracellularly.In a process which is dependent on energy and temperature, Fetransferrin or Fe₂ transferrin is bound to the receptor on the cellmembrane. Then the complex is absorbed into a vesicle, referred to asendosome or receptosome. This is combined with another vesicle having apH of <5.5; the resulting acidification causes the iron to be releasedby the transferrin. The apotransferrin receptor complex is thentransported back to the cell membrane, where the neutral pH causes theapotransferrin to be released from the receptor into the surroundingmedium. There are indications that the recycling, the functioning ofwhich is based on the affinity of the receptor for apo- or irontransferrin, which varies at acid and neutral pH values, takes placethrough vesicles of the Golgi apparatus.

At a molecular level, the initiation of the transferrin cycle has notyet been explained; there are merely indications regarding some aspects,e.g. the possible role of phosphorylation (Huebers et al., 1987).

With the present invention it has been possible for the first time tomake use of this active transporting system in order to convey into thecell nucleic acids, the absorption of which runs into difficulties.

The present invention thus relates to new protein-polycation conjugateswhich are capable of forming complexes with substances having anaffinity for polycations, particularly nucleic acids or nucleic acidanalogues, and these complexes are absorbed into the cell by means ofreceptor-mediated endocytosis, the protein content of the conjugatebeing transferrin.

It has been found, surprisingly that nucleic acids can be efficientlytransported into the cell whilst maintaining their inhibitory activity,using the conjugates according to the invention.

The word "transferring" in accordance with this invention relates toboth the natural transferrins and also to transferrin modificationswhich are bound by the receptor and transported into the cell (suchmodifications may consist, for example, of a change in the amino acidsor a shortening of the molecule to the fraction which is responsible forreceptor binding).

The molar ratio of transferrin to polycation is preferably 10:1 to 1:4,whilst it should be borne in mind that aggregates may be formed.However, this ratio may if necessary be within wider limits, providedthat it satisfies the condition that complexing of the nucleic acid oracids takes place and it is ensured that the complex formed-will bebound by the transferrin receptor and conveyed into the cell; this caneasily be checked by simple experiments carried out from one case to thenext.

The ratio chosen will depend particularly on the size of the polycationmolecule and the number and distribution of the positively chargedgroupings, these criteria being matched to the size and structure of thenucleic acid or acids to be transported and to any modificationsthereto. The polycations may be identical or different.

The following compounds may be used as polycations:

a) Protamines: these are small (MW up to about 8000) strongly basicproteins, the positively charged amino acid groups of which (especiallyarginines) are usually arranged in groups and neutralise the negativecharges of nucleic acids because of their polycationic nature (Warrantet al., 1978). The protamines which may be used within the scope of thepresent invention may be of natural origin or produced by recombinantmethods, whilst multiple copies may be produced or modifications may bemade in the molecular size and amino acid sequence. Correspondingcompounds may also be chemically synthesised. When an artificialprotamine is synthesised the procedure used may consist, for example, inreplacing amino acid residues, which have functions in the naturalprotamine which are undesirable for the transporting function (e.g. thecondensation of DNA) with other suitable amino acids, and/or at one endproviding an amino acid (e.g. cysteine) which will enable the desiredconjugation with transferrin.

b) Histones: these are small DNA-binding proteins present in thechromatin, having a high proportion of positively charged amino acids(lysine and arginine) which enable them to bind to DNA independently ofthe nucleotide sequence and fold it into nucleosomes, the arginine-richhistones H3 and H4 being particularly suitable (Felsenfeld, 1978). Asfor the preparation and modifications thereof, the remarks made above inrelation to protamines apply here as well.

c) Synthetic polypeptides such as homologous polypeptides (polylysine,polyarginine) or heterologous polypeptides (consisting of two or morerepresentatives of positively charged amino acids).

d) Non-peptide cations such as polyethyleneimines.

The size of the polycations is preferably selected so that the sum ofthe positive charges is about 20 to 500 and this is matched to thenucleic acid which is to be transported.

The transferrin-polycation conjugates according to the invention may beproduced chemically or, if the polycation is a polypeptide, by therecombinant method. Coupling by the chemical method can be carried outin a manner known per se for the coupling of peptides and if necessarythe individual components may be provided with linker substances beforethe coupling reaction (this procedure is necessary when there is nofunctional group suitable for coupling available at the outset, such asa mercapto or alcohol group). The linker substances are bifunctionalcompounds which are first reacted with functional groups of theindividual components, after which coupling of the modified individualcomponents is carried out.

Depending on the desired properties of the conjugates, particularly thedesired stability thereof, coupling may be carried out by means of

a) disulphide bridges, which can be cleaved again under reductiveconditions (e.g. using succinimidyl pyridyl dithiopropionate (Jung etal., 1981)).

b) Using compounds which are largely stable under biological conditions(e.g. thioethers, by reacting maleimido linkers with sulfhydryl groupsof the linker bound to the second component).

c) Using bridges which are unstable under biological conditions, e.g.ester bonds, or using acetal or ketal bonds which are unstable underweakly acidic conditions.

The production of the conjugates according to the invention by therecombinant method offers the advantage of producing precisely defined,uniform compounds, whereas chemical coupling produces conjugate mixtureswhich then have to be separated.

The recombinant preparation of the conjugates according to the inventioncan be carried out using methods known for the production of chimericpolypeptides. The polycationic peptides may vary in terms of their sizeand amino acid sequence. Production by genetic engineering also has theadvantage of allowing the transferrin part of the conjugate to bemodified, by increasing the ability to bind to the receptor, by suitablemutations, for example, or by shortening the transferrin fraction to thepart of the molecule which is responsible for the binding to thereceptor. It is particularly expedient for the recombinant preparationof the conjugates according to the invention to use a vector whichcontains the sequence coding for the transferrin part as well as apolylinker into which the required sequence coding for the polycationicpeptide is inserted. In this way, a set of express plasmids can beobtained, of which the plasmid containing the desired sequence can beused as necessary in order to express the conjugate according to theinvention.

The nucleic acids which are to be transported into the cell may be DNAsor RNAs, with no restrictions as to the nucleotide sequence. The nucleicacids may be modified, provided that this modification does not affectthe polyanionic nature of the nucleic acids; these modificationsinclude, for example, the substitution of the phosphodiester group byphosphorothioates or the use of nucleoside analogues.

Nucleic acids which may be used within the scope of the presentinvention include particularly those which are to be transported intothe cell for the purpose of inhibiting specific gene sequences. Theseinclude antisense oligonucleotides and ribozymes, optionally togetherwith a carrier nucleic acid. With regard to the size of the nucleicacids the invention again permits a wide range of uses. There is nolower limit brought about by the transporting system according to theinvention; any lower limit which might arise would be for reasonsspecific to the application, e.g. because antisense oligonucleotideswith less than about 10 nucleotides would hardly be suitable owing totheir lack of specificity. Using the conjugates according to theinvention it is also possible to convey plasmids into the cell, thesmaller plasmids which are used as carrier nucleic acids (e.g.retroviral vectors with up to 5000 bp) being of particular practicaluse. It is also possible to convey different nucleic acids into the cellat the same time using the conjugates according to the invention.

A further advantage of the present invention is the fact that there arepolymorphisms for transferrin and the receptor which can be exploitedfor the deliberate transporting of inhibiting nucleic acids intospecific cells.

Within the scope of the present invention it has been possible todemonstrate that transferrin-polycation conjugates can be efficientlyabsorbed into living cells and internalised. The conjugates or complexesaccording to the invention are not harmful to cell growth. This meansthat they can be administered repeatedly and thus ensures a constantlyhigh expression of genes inserted into the cell.

It has also been possible to show that the polycation-transferrinconjugates can functionally replace the native transferrin-iron complex.The fact that the transferrin-polycation/DNA complexes are absorbedthrough the cell by means of the transferrin receptor has been confirmedusing the luciferase gene as DNA component. It has been shown thatnative transferrin efficiently displaces the transferrin-polycation/DNAcomplex, and this has been measured by the reduction in the luciferaseactivity in the cell.

The experiments carried out within the scope of this invention have alsodemonstrated that a tRNA ribozyme gene (the ribozyme being directedagainst a V-erbB sequence) can be introduced into erbB-transformedchicken cells using a conjugate according to the invention(polylysine-transferrin) and can attenuate the transforming activity ofthe oncogene. This result is all the more significant as only a smallamount of ribosome gene was used in these experiments.

The ratio of nucleic acid to conjugate can vary within a wide range, andit is not absolutely necessary to neutralise all the charges of thenucleic acid. This ratio will have to be adjusted for each individualcase depending on criteria such as the size and structure of the nucleicacid which is to be transported, the size of the polycation and thenumber and distribution of its charges, so as to achieve a ratio oftransportability and biological activity of the nucleic acid which isfavourable to the particular application. This ratio can first of all beadjusted coarsely, for example by using the delay in the speed ofmigration of the DNA in a gel (e.g. using the mobility shift on anagarose gel) or by density gradient centrifugation. Once thisprovisional ratio has been obtained, it may be expedient to carry outtransporting tests with the radioactively labelled complex with respectto the maximum available activity of the nucleic acid in the cell andthen reduce the proportion of conjugate if necessary so that theremaining negative charges of the nucleic acid are not an obstacle totransportation into the cell.

The preparation of the transferrin-polycation/nucleic acid complexes,which are also a subject of the invention, can be carried out usingmethods known Per se for the complexing of polyionic compounds. Onepossible way of avoiding uncontrolled aggregation or precipitation is tomix the two components together first of all at a high (about 1 molar)concentration of common salt and subsequently to adjust to physiologicalsaline concentration by dialysis or dilution. Preferably, theconcentrations of DNA and conjugate used in the complex forming reactionare not too high (more than 100 μg/ml), to ensure that the complexes arenot precipitated.

The preferred nucleic acid component of thetransferrin-polycation-nucleic acid complex according to the inventionis antisense-DNA, antisense-RNA or a ribozyme or the gene coding for it.When ribozymes and antisense-RNAs are used it is particularlyadvantageous to use the genes coding for these RNAs which inhibit thefunction of RNA, preferably together with a carrier gene. By introducingthe gene into the cell, a substantial amplification of the RNA isensured, as against the importing of RNA as such, and consequently asufficient amount to inhibit the biological reaction is ensured.Particularly suitable carrier genes are the transcription units, e.g.tRNA genes, required for transcription by polymerase III. Ribozymegenes, for example, may be inserted into them in such a way that whenthe transcription is carried out the ribozyme is part of a compactpolymerase III transcript. Using the transporting system according tothe present invention it is possible to intensify the activity of thesegenetic units, by guaranteeing an increased initial concentration of thegene in the cell.

The invention further relates to a process for introducing nucleic acidor acids into human or animal cells, preferably forming a complex whichis soluble under physiological conditions.

The invention further relates to pharmaceutical preparations containingas the active component a nucleic acid which specifically inhibits agene, complexed with a transferrin-polycation conjugate, e.g. in theform of a lyophilisate. Such pharmaceutical preparations may be used toinhibit pathogenic viruses such as HIV or related retroviruses,oncogenes or other key genes which control growth and/or differentiationof cells, e.g. the c-fos gene or the c-myc gene, together with antisenseoligonucleotides, antisense oligonucleotide analogues or ribozymes orthe DNAs coding for them, optionally together with a carrier nucleicacid, in humans or animals.

Another field of use is in fighting diseases by inhibiting theproduction of undesirable gene products, e.g. the major plaque proteinwhich occurs in Alzheimer's disease or proteins which cause autoimmunediseases.

The invention is illustrated by means of the Examples which follow.

EXAMPLE 1

Preparation of transferrin-polylysine 90 conjugates

Coupling was carried out using methods known from the literature (Junget al., (1981)) by introducing disulphide bridges after modificationwith succinimidyl-pyridyl dithiopropionate.

a) 3-(2-Pyridyldithio)propionate-modified transferrin

6 ml of a solution, gel-filtered over Sephadex G-25, of 120 mg (1.5μmol) of transferrin (from chicken albumin, Sigma, Conalbumin Type I,iron-free) in 3 ml of 0.1M sodium phosphate buffer (pH 7.8) were mixedwith 200 μl of 15 mM ethanolic solution of succinimidyl3-(2-pyridyldithio)propionate (3 μM, SPDP, Pharmacia) with vigorousshaking and the mixture was left to react for 1 hour at ambienttemperature with occasional shaking. Low molecular reaction products andtraces of reagent were removed using a gel column (Sephadex G-25, 14×180mm, 0.1M sodium phosphate buffer pH 7.8) and 7 ml of the productfraction were obtained; the content of pyridyl dithiopropionate residuesbound to transferrin was determined by means of one aliquot, afterreduction with dithiothreitol, by photometric measurement of thequantity of pyridin-2-thione released and the result was 2.6 μmol. Humantransferrin (Sigma, iron-free) was modified in exactly the same way.

b) Mercaptopropionate-modified polylysine 90 (pL 90)

A solution of 18 mg (about 1.0 μmol) of poly(L)lysine-hydrobromide(Sigma, fluoresceinisothio-cyanate (=FITC)-labelled, molecular weight ofabout 18,000--corresponding to an average degree of polymerisation ofabout 90) in 3 ml of 0.1M sodium phosphate (pH 7.8) was filtered overSephadex G-25 (the fluorescent labelling was carried out in sodiumbicarbonate buffer pH 9 for 3 hours). The polylysine solution wasdiluted with water to 7 ml, combined with 270 μl of a 15 mM ethanolicsolution of SPDP with thorough shaking and then left to react for 1 hourin the dark at ambient temperature and with occasional shaking. Afterthe addition of 0.5 ml of 1M sodium acetate buffer (pH 5.0) the mixturewas filtered over Sephadex G-25 to separate off any lower molecularsubstances (eluant: 20 mM sodium acetate buffer pH 5.0). The productfraction (ninhydrin staining, fluorescence) was evaporated down invacuo, adjusted to pH about 7 with buffer, a solution of 23 mg (150μmol) of dithiothreitol in 200 μl of water was added and the mixture wasleft to stand for 1 hour in the dark under argon at ambient temperature.Excess reducing agent was separated off by further gel filtration(Sephadex G-25, 14×130 mm column, 10 mm sodium acetate buffer pH 5.0)and 3.5 ml of product solution of fluorescently labelled polylysine wereobtained, containing 3.8 μmol of mercapto groups (photometricdetermination using Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoicacid).

c) Transferrin-polylysine conjugates

The solution of modified transferrin obtained in a) (7 ml in 0.1M sodiumphosphate buffer pH 7.8, about 1.5 μmol transferrin with about 2.6 μmolpyridyl dithiopropionate residues) was rinsed with argon; 2.0 ml of thesolution of mercapto-modified polylysine obtained in b) (in 10 mm sodiumacetate buffer pH 5.0, corresponding to about 0.6 μmol of polylysinewith about 2.2 μmol of mercapto groups) were added, the mixture wasrinsed with argon, shaken and left to react for 18 hours at ambienttemperature in the dark and under argon. The reaction mixture wasdiluted with water to 14 ml and separated by ion exchange chromatography(Pharmacia Mono S column HR 10/10, gradient elution, buffer A: 50 mMHEPES pH 7.9, buffer B: A+3M sodium chloride, 0.5 ml/min, FIG. 1).Non-conjugated transferrin was eluted at the start, product fractions atabout 0.66-1.5M sodium chloride.

Averaged over all the fractions, conjugates were obtained containing aratio of transferrin to polylysine of 1.3:1.

The conjugated products (ninhydrin staining, in UV at 280 nm proteinabsorption, and fluorescence measurement of FITC-labelled polylysine at495 nm) were collected in 6 fractions each containing about 10 mg oftransferrin. The fractions were first dialysed against a 100 mmiron(III)citrate solution (adjusted to pH 7.8 with sodium hydrogencarbonate) and then twice more with 1 mM HEPES buffer (pH 7.5).

Sodium dodecylsulphate gel electrophoresis (10% SDS, 8% polyacrylamide),see FIG. 2, showed an approximately equal content of transferrin in all6 fractions after pretreatment with 2-mercaptoethanol (FIG. 2A), whereasin the unreduced samples there were no visible bands for freetransferrin, only less widely migrating conjugates (FIG. 2B, T=untreatedtransferrin; 1-6=conjugate fractions 1-6).

EXAMPLE 2 Preparation of transferrin-polylysine 270 andtransferrin-polylysine 450 conjugates (pL270 and pL450)

a) Modified transferrin was produced analogously to Example 1 a)

b) Preparation of modified polylysine 270 and polylysine 450

A gel-filtered solution of 0.33 μmol polylysine 270 (with an averagedegree of polymerisation of 270 lysine groups, with or withoutfluorescent labelling; corresponding to 19 mg of hydrobromide salt) in1.2 ml of 75 mM sodium acetate buffer was adjusted to pH 8.5 by theaddition of sodium carbonate buffer. 182 μl of a 15 mM ethanolicsolution of SPDP (1.9 μmol) was added with vigorous stirring. One hourlater, 200 μl 1M sodium acetate pH 5 were added; after gel filtrationwith 20 mM sodium acetate, a solution was obtained containing 0.27 μmolof polylysine 270 with 1.3 μmol mercapto groups (4.8 linkers perpolylysine chain). Analogously, 0.20 μmol of polylysine 450 (with anaverage degree of polymerisation of 450 lysine groups) were modifiedwith 2.25 μmol of SPDP, obtaining a product of 0.19 μmol polylysine 450with 2.1 μmol mercapto groups (11 linkers per polylysine chain).Analogously to Example 1 b), the dithiopyridine groups were reduced withdithiothreitol, in order to obtain the free sulfhydryl components.

c) Preparation of transferrin-polylysine conjugates

Transferrin-polylysine 270 conjugates were prepared by mixing 1.0 μmolof modified transferrin in 100 mM phosphate buffer, pH 7.8, with 0.14μmol of modified polylysine 270 (in 20 mM sodium acetate buffer) withthe exclusion of oxygen in an argon atmosphere. After 18 hours atambient temperature the reaction mixture was diluted with water to avolume of 10 ml and separated by cation exchange chromatography(Pharmacia Mono S column HR 10/10; gradient elution, buffer A: 50 mMHEPES pH 7.9; buffer B: A+3M sodium chloride; UV absorption at 280 nmand fluorescence measurement, excitation 480 nm, emission 530 nm). Theexcess of non-coupled transferrin was eluted first; the productfractions were eluted at between 30% and 50% gradient B and pooled in 3conjugate fractions (molar ratios of transferrin to polylysine: pool A:5.5 to 1; pool B: 3.4 to 1; pool C: 1.8 to 1). The conjugates wereobtained in an average yield of 0.23 μmol transferrin with 0.075 μmol ofpolylysine 270.

Transferrin-polylysine 450 conjugates were prepared in a similar manner,starting from 1.2 μmol of modified transferrin according to Example 1 a)(in 20 mM HEPES pH 7.9 containing 80 mM sodium chloride) and 71 nmol ofmercapto-modified polylysine 450 according to Example 2 b) in acetatebuffer.

The reaction mixture was purified by gel permeation chromatography(Pharmacia Superose 12 column, 1M guanidine chloride pH 7.3) and afterdialysis (20 mM HEPES pH 7.3, containing 100 mM sodium chloride) yieldedtransferrin-polylysine-conjugates containing 0.40 μmol of transferrinwith 38 nmol of polylysine 450.

Iron was incorporated by adding 6-12 μl of 100 mM iron citrate buffer(containing sodium bicarbonate adjusted to pH 7.8) to the samples, permg of transferrin fraction.

EXAMPLE 3

a) Preparation of transferrin-protamine conjugates

Modified transferrin was prepared as in Example 1 a).

b) Preparation of 3-mercaptopropionate-modified protamine

To a solution of 20 mg (3 μmol) of protamine trifluoracetate salt(prepared by ion exchange chromatography from salmon protamine(=salmin)-sulphate, Sigma) in 2 ml of DMSO and 0.4 ml of isopropanol,containing 2.6 μl (15 μmol) of ethyl diisopropylamine, was added asolution of 30 μmol of SPDP in 250 μl of isopropanol and 250 μl of DMSOin several batches over a period of one hour. After 3.5 hours at ambienttemperature the solution was evaporated down under high vacuum and takenup in 0.5% acetic acid containing 10% methanol. Gel filtration (SephadexG10; 0.5% acetic acid with 10% methanol) yielded, after lyophilisation,16 mg (2.5 μmol of protamine acetate salt, modified with 2.5 μmol ofdithiopyridine linker. Reduction of 1.75 μmol of protamine (containing1.75 μmol of linker) with 16 mg of dithiothreitol in sodium bicarbonatebuffer, pH 7.5, for 1 hour under argon, followed by adjustment of the pHto 5.2 and gel filtration over Sephadex G10 with 20 mM sodium acetatebuffer, pH 5.2, yielded a protamine solution modified with 1.6 μmol ofmercaptopropionate linker.

c) Preparation of transferrin-protamine conjugates

The reaction of the protamine solution obtained in b) (1.6 μmol linker)with 1.34 μmol of transferrin (modified with 3.1 μmol of dithiopyridinelinker) and subsequent purification by cation exchange chromatography asdescribed for transferrin-polylysine conjugates, yielded four productfractions A-D eluted one after the other, containing 90, 320, 240 and120 nMol, respectively, of modified transferrin with increasing amountsof protamine (determined by SDS gel electrophoresis; 10% SDS, 8%polyacrylamide, Coomassie blue staining). FIG. 3 shows the results ofthe SDS gel electrophoresis. The Tfprot conjugate fractions A-D moreslowly migrating bands (a), whereas in β-mercaptoethanol-reduced samples(b) only the transferrin band was visible. Dialysis and theincorporation of iron were carried out as described for thetransferrin-polylysine conjugates TfpL270 and TfpL450 in Example 2.

EXAMPLE 4 Preparation of complexes of transferrin-polycation conjugateswith DNA

The complexes were prepared by mixing dilute solutions of DNA (30 μg/mlor less) with the transferrin-polycation conjugates. In order to preventprecipitation of the DNA complexes, phosphate-free buffer was used(phosphates reduce the solubility of the conjugates). The binding of theDNA to the polycation conjugates under physiological ionic conditionswas confirmed by a gel mobility shift assay using lambda DNA ³²P-labelled at the 3'-end, cut with EcoR 1/Hind III (FIG. 4). To eachsample of 1 μl (35 ng) of DNA were added 3 μml of a 100 mM HEPES bufferpH 7.9, containing 1M sodium chloride, and the samples were mixed withincreasing amounts (10 ng to 1000 ng) of transferrin conjugates in 11 μlof aqueous solution, resulting in a final concentration of sodiumchloride of 200 mM. Electrophoresis on a 1% agarose gel with 1×TAEeluting buffer was carried out at 50 Volts (45 mA) for 2.5 hours; thegel was dried, followed by autoradiography for 2 hours at -80° C. usingan XAR film (Kodak).

EXAMPLE 5 Transporting of transferrin-polylysine conjugates into livingcells

In order to demonstrate that the transferrin-polylysine conjugatesdescribed in Example 1 are efficiently absorbed into livingerythroblasts, FITC-labelled conjugates were used. It is known (Schmidtet al, 1986) that FITC-labelled transferrin was detectable in vesiclesinside the cell (when examined under a fluorescence microscope) aftersome hours' incubation with erythroblasts from which transferrin hadpreviously been removed.

In the present Example, erythroblasts (transformed by an EGF-receptorretrovirus, Khazaie et al, 1988) were incubated for 18 hours in atransferrin-free differentiating medium (composition in Zenke et al,1988) at 37° C. (cell concentration 1.5×10⁶ /ml). After addition of thevarious transferrin-polylysine conjugates (or, as a control, thecorresponding amount of sterile twice distilled water), the cells wereincubated at 37° C. in the presence of 10 ng/ml EGF in order to maintainthe transformed state. After 24 and 48 hours, about 5×10⁵ cells wereremoved, washed once in phosphate-buffered physiological saline (PBS; pH7.2), fixed with 50 times the volume of a mixture of 3.7% formaldehydeand 0.02% glutaraldehyde in PBS (10 minutes, 40° C.), washed once inPBS, embedded in Elvanol and examined under a fluorescence microscope(Zeiss Axiophot, Narrow Band FITC and TRITC activation). At the sametime, the growth rate of the cells was determined in other aliquots ofthe various mixtures. 100 μl cell suspension were taken and theincorporation of ³ H-thymidine (8 μCi/ml, 2 hours) was determined asdescribed in Leutz et al, 1984. FIG. 5 shows that the erythroblastsincubated with transferrin-polylysine show 2 to 10 strongly fluorescingvesicles after 24 hours, which cannot be detected in the controls. TableA shows that, with the exception of fraction 6, all the conjugates havebeen absorbed by virtually all the cells.

FIG. 5 shows fluorescence images of chicken erythroblasts which havebeen incubated for 24 hours without (A) or with FITC-labelledtransferrin-polylysine conjugates (B,C). When they are activated withblue light (B, in order to detect FITC), significantly more fluorescingvesicles can be detected in each cell. The specificity of thisfluorescence is shown by the fact that the vesicle fluorescence does notoccur when activated with green light (C: at which a similarnon-specific fluorescence of the cells can be seen as in A) (C).

The fact that the cells grow equally rapidly in all the samples (asmeasured by the incorporation of tritiated thymidine (³ H TdR), Table A)shows that the cells are not dimensioned by the polylysine constructsand consequently non-specific uptake (e.g. through cell membranes whichhave become permeable) can be ruled out.

EXAMPLE 6

The objective of the tests carried out in this Example was to show thatthe transferrin-polylysine conjugates used here are used by the cell inthe same way as native transferrin, i.e. they pass through the normaltransferrin cycle with similar efficiency. Erythroblasts which can beinduced to mature into normal erythrocytes by "switching off" thetransforming oncogene are particularly suitable as a test system forthis purpose (Beug et al, 1982). The literature shows that for normalmaturation such cells require high concentrations of transferrin-ironcomplex (100 to 200 μg/ml, 3 times lower concentrations prevent thecells from maturing and will result in the death of the cells afterseveral days (Kowenz et al, 1986)). It has also been shown (Schmidt etal 1986) that recycling, i.e. the reuse of transferrin receptors andhence a transferrin cycle proceeding at optimum speed are indispensiblefor normal in vitro differentiation.

Erythroblasts (transformed by the EGF-receptor retrovirus) were inducedto differentiate by the removal of EGF and the addition of an optimumamount of partially purified chicken erythropoietin (Kowenz et al.,1986, free from transferrint. Incubation was carried out at a cellconcentration of 1×10⁶ /ml in transferrin-free differentiating medium at42° C. and 5% CO₂. A t the start of incubation, either nativetransferrin-iron complex (Sigma, 100 μg/ml) was added or theiron-saturated transferrin-polylysine conjugates were added(concentration again 100 μg/ml). The growth and maturity of the cellswere analysed are 24 and 48 hours by the following methods:

1. by determining the number of cells (using a Coulter Counter, ModelZM, Beug et al, 1984)

2. by recording cell size distributions (using a Coulter ChannelyzerModel 256) and

3. by photometric determination of the haemoglobin content of the cells(Kowenz et al., 1986).

In addition, aliquots of the mixtures were centrifuged after 72 hours ina cytocentrifuge (Shandon) on an object carrier and subjected tohistochemical investigation t o detect haemoglobin (staining withneutral benzidine plus Diff-Quik rapid staining for blood cells, Beug etal 1982).

The results in Table B clearly show that cells which were induced todifferentiate in the presence of the polylysine-transferrin conjugatesfractions 1 to 5 mature just as efficiently and as fast as those whichwere incubated with native transferrin-iron. The cells in thetransferrin-free controls, on the other hand, showed a much slower cellgrowth and accumulated only small amounts of haemoglobin. Investigationof cell phenotype on stained cytospin preparations showed that the cellsincubated with polylysine-transferrin conjugates were matured into latereticulocytes (late reticulocytes, Beug et al., 1982) in just the sameway as those which had been treated with native transferrin, whereas thecells incubated without transferrin constituted a mixture ofdisintegrated and immature cells resembling erythroblasts (Schmidt etal, 1986). Only the cells treated with transferrin-polylysine fraction 6showed a lower haemoglobin content and a higher percentage of immaturecells (Table B). This shows that fraction 6 conjugated with aparticularly large amount of polylysine operates less well in thetransferrin cycle. At the same time, this result indicates thesensitivity of the test method.

EXAMPLE 7

Just as in Example 6, various transferrin-polylysine conjugates andtransferrin-protamine conjugates were examined for their ability tofunctionally replace the native transferrin-iron complex in thematuration of chicken erythroblasts into erythrocytes.

It has already been shown that terminally differentiating chickenerythroblasts demand an optimally functioning transferrin cycle; i.e.without transferrin or if the transferrin receptor recycling isinhibited, the cells die off (Kowenz et al., 1986; Schmidt et al.,1986). Since the partially purified chicken erythropoietin normally usedstill contains transferrin, EPO was replaced by a transferrin-free,partially purified erythroid growth factor in order to permit erythroiddifferentiation (REV factor; Kowenz et al., 1986; Beug et al., 1982): astarget cells, erythroblasts which had been transformed with a retroviruscontaining the human epidermal growth factor receptor (EGFR) togetherwith a temperature-sensitive v-myb oncogen were replicated in CFU-Emedium (Radke et al., 1982) in the presence of 20 ng/ml of EGF. Thesecells are activated to replicate abnormally by EGF, whilst thewithdrawal of the growth factor EGF and the simultaneous addition of REVfactor causes the cells to enter into normal differentiation. Afterbeing washed twice in transferrin-free medium the cells were transferredinto transferrin-free medium and varying quantities of iron-saturatedtransferrin or transferrin-polycation conjugates were added (before orafter being complexed with plasmid DNA). After 1, 2 and 3 days'incubation at 42° C. the differentiating state of the cells wasdetermined by cytocentrifugation and histochemical staining or byquantitative haemoglobin measurement.

The results of these tests are shown in FIG. 6 or Table C.

The cells (1×10⁶ /ml) were added to conalbumin-free differentiatingmedium (Zenke et al., 1988), supplemented by 1 μg/ml of insulin and REVfactor at an optimum concentration (Kowenz et al., 1986; dilution1:5,000), once without additives (triangles), once with iron-saturatedconalbumin (circles) and once with iron-saturated TfpL 270 conjugates(squares) (100 μg/ml in each case); in 14 mm dishes. After incubationfor 24 and 48 hours, the haemoglobin content was photometricallydetermined in 100 μl aliquots. The shaded area shows the haemoglobincontent of cells grown without transferrin (average from 4 measurements;FIG. 6A).

In order to analysis the erythroid differentiation as a function of theconcentration of transferrin or transferrin-polylysine, the cells wereplaced in medium containing the specified amounts of iron-saturatedconalbumin (open circles), TfpL 90 (open squares) or TfpL 270 (solidsquares) as described above and after 2 days the haemoglobin content wasdetermined photometrically (FIG. 6B).

Table C

The erythroid differentiation was monitored by photometric haemoglobinmeasurement (see FIG. 6), by counting in a Coulter counter or bycytocentrifugation and subsequent neutral benzidine staining (todetermine the haemoglobin) plus histological dyes (Diff Quik; Beug etal., 1982b). The final concentrations of transferrin andtransferrin.conjugates in test 1 were 60 μg/ml; in tests 2 and 3 theywere 100 μg/ml. The DNA concentration in test 2 was 10 μg/ml. Theproportion of disintegrated cells, mature cells (LR: late reticulocytes;E: erythrocytes) and immature cells (Eb1) was determined using themethods described by Beug et al., 1982b and Schmidt et al., 1986. Theresults obtained show that two different transferrin-polylysineconjugates (TfpL90 or TfpL270) as well as the transferrin-protamineconjugate are capable of functionally replacing native transferrin, byensuring the rapid transfer of iron into differentiating red cells, thespecific activity thereof being 1.5 to 2 times lower (cf. FIG. 6). Thecomplexing of DNA with transferrin-polylysine 270 and transferrinprotein did not materially alter the biological activity of theconjugates. In a control experiment it was established that, whenpolylysine or protamine is added, mixed with a suitable quantity of ironcitrate instead of the transferrin conjugates, the cells were unable todifferentiate and died off, just like the cells in the comparisonsamples which had been incubated without transferrin.

All in all, the tests according to Examples 6 and 7 have shown that bothtypes of polycation-transferrin conjugates transported iron onlyslightly less efficiently than natural transferrin.

EXAMPLE 8

Polylysine-transferrin conjugates make it possible for DNA to beabsorbed into chicken erythroblasts.

The present Example was intended to investigate whether DNA of a sizecorresponding to that of tDNA ribozymes (see FIG. 7) is capable of beingefficiently transported into the interior of the cell bytransferrin-polylysine conjugates. In the present Example, tDNA with aninsert of the sequence

CGTTAACAAGCTAACGTTGAGGGGCATGATATCGGGCCCCGGGCAATTGTTCGATTGCAACTCCCCGTACTATAGC

molecular weight about 300,000 was used, terminally labelled with gamma32P ATP (Maniatis). About 0.3 μg of this DNA, dissolved in 20 μl of TEbuffer were mixed either with 10 μg of native transferrin, with 10 μg oftransferrin-polylysine conjugate fraction 3, in each case dissolved in50 μl of twice distilled water plus 400 μg/ml of bovine serum albumin(Beug H., et al., 1982) or with 50 μl of this solvent withouttransferrin. The DNA protein mixtures were each added to 2 ml oftransferrin-free differentiating medium, 4×10⁶ chicken erythroblastswere added (which had previously been transformed with an EGF receptorretrovirus and preincubated for 18 hours in transferrin-free medium inthe presence of EGF (Khazaie K., et al., 1988) and the mixtures wereincubated for 8 hours at 37° C. and 5% CO₂. Then the cells werecentrifuged off, the supernatant was removed and the cells were washed 3times in transferrin-free medium. Cell sediment and culture medium weretaken up in 1% SDS, 1 mg/ml of proteinase K, 300 mM NaCl, 20 mM tris pH8.0, 10 mM EDTA (PK/SDS buffer), incubated for 30 minutes at 37° C.,extracted with phenol/chloroform, and the DNA isolated by ethanolprecipitation. Isolated DNA with a radioactivity of 2000 cpm in all wereseparated on a non-denaturing 3.5% acrylamide gel (TBE, Maniatis) andthe DNA was detected by autoradiography. It was shown that, in the cellsample treated with transferrin-polylysine, approximately 5 to 10 timesmore DNA had been absorbed by the cells than in the control samples withnative transferrin.

EXAMPLE 9

Polylysine-transferrin conjugates make it possible for plasmid-DNA to beabsorbed into and expressed in chicken erythroblasts.

In these tests, plasmid-DNA containing the Photinus pyralis luciferasegene as reporter gene was used to investigate gene transfer andexpression. For this purpose, pRSVluc plasmid DNA (De Wet, J. R., etal., 1987) was prepared using the Triton-X lysis standard method(Maniatis), followed by CsCl/EtBr equilibrium density gradientcentrifugation, decolorising with butanol-1 and dialysis with 10 mmTris/HCl, pH 7.5, 1 mm EDTA. In a typical complex forming reaction, 10μg of transferrin-polylysine or transferrin-protamine conjugates wereslowly added, with careful stirring, to 3 μg of pRSVluc plasmid DNAcontained in 250 μl of 0.3M NaCl (it was established that theseconditions are adhered to, up to 100 μg of transferrin-polycationconjugate and 30 μg of plasmid-DNA can be used in a final volume of 500μl without precipitation of the conjugate/DNA complexes). After 30minutes at ambient temperature, the complexes were added directly to5-10×10⁶ HD3 cells (0.5-1×10⁶ cells per ml, EBM+H medium (Beug et al.,1982a; 37° C., 5% CO₂) and the mixtures were incubated for 16 to 48hours (the cell line used was the ts-v-erbB transformed chickenerythroblast cell line HD3). The cells were harvested (5 min at 1500×g,4° C., washed twice with phosphate-buffered saline (PBS) and taken up in100 μl of 0.25M tris/HCl, pH 7.5. Cell extracts were prepared by threecycles of freezing and thawing, followed by high-speed centrifugation(15 min, 18,500×g, 4° C.). Aliquots of these cell extracts were examinedfor the presence of luciferase enzyme activity (De Wet, J. R., et al.,1987). The bioluminescence was measured using clinilumate (Berthold,Wildbach, BRD). It was established that the presence of thetransferrin-polycation/DNA complexes in the culture medium does not haveany harmful effects on cell growth or replication. As can be seen fromFIG. 8, maximum luciferase activity was achieved when using 3 μg ofDNA/10 μg of TfpL and 0.3-1 μg of DNA/Tfprot. Assuming that all theconjugate/DNA complexes formed were identical, this corresponds to amolar ratio of 25 or 75 conjugate molecules per molecule of plasmid DNA.It can be concluded from this that the DNA in the complex is entirelycovered by the conjugate molecules, and indeed at a conjugate/DNA ratiowhich obviously guarantees electroneutrality (calculated on the basis ofthe positive charges in the polycation which are necessary in order toneutralise the negative charges of the phosphate groups in the DNA).This assumption agrees with the observation that, compared withtransferrin-polylysine, three times more transferrin/protamine, lessstrongly positively charged, is required for optimum complex forming andgene transfer. This assumption also accords with the results for theconjugate/DNA ratio obtained in Example 4 required for efficient complexformation.

The sensitivity of this gene transfer system was determined using aTfpL/DNA ratio which had been adjusted to the optimum for complexforming. The results of these tests are shown in FIG. 9: less than 1 ngof plasmid DNA coding for luciferase still shows a detectable signal.The use of more than 2 μg of plasmid DNA, complexed with 6 μg of TfpL or20 μg of Tfprot, does not result in any further increase in luciferaseactivity, presumably because the system is saturated. It was also foundthat no special concentration of salt or ions is needed for the complexforming, since TfpL/DNA complexes formed at various salt concentrations(0, 20, 50 100, 200 mM NaCl) prove to be equally effective in genetransfer experiments. (FIG. 8 and FIG. 9: circles indicate Tfprot,squares indicate TfpL). It was possible to demonstrate that theabsorption of transferrin-polycation/DNA complexes into the cells waseffected via the transferrin receptor. First of all, as illustrated inFIG. 10A, it was found that the luciferase activity achieved by TfpL-DNAcomplexes is at least 100 times higher than the activity measured forpL-DNA complexes. A comparison test shows that a mixture of polylysineand transferrin alone did not facilitate the uptake of plasmid DNA. Inanother test, an excess of native transferrin was added to a constantquantity of TfpL-DNA complex. FIG. 10B shows that free transferrin inthe medium efficiently competes for the DNA uptake mediated by TfpL,resulting in a reduction in the luciferase enzyme activity. From this itcan be concluded that the uptake of the TfpL-DNA complexes by the cellis effected via the transferrin receptor.

EXAMPLE 10

In preliminary tests it was established, by the transfection of chickenfibroblasts with erbB cut DNA that the erbB cut ribozyme-tDNA isexpressed in chicken cells.

This Example shows that tDNA ribozymes directed against the erbBoncogene can be introduced into erbB-transformed chicken erythroblastswith the aid of polylysine-transferrin conjugates and can weaken thetransforming activity of the oncogene.

Two tRNA ribozyme genes, directed against the translation initiationregion of erbB, were constructed (see FIGS. 7 and 11). About 100 μg ofeach plasmid containing the gene were digested in EcoRI in order to freethe tRNA ribozyme gene on a 325 bp fragment.

The digestion products were terminally labelled by means of klenowfragment and purified by gel electrophoresis using a 2% agarose/TBE gel.The vector fragment and the tRNA-ribozyme gene fragments were located bystaining with ethidium bromide, cut out and recovered by electroelution,extraction with phenol/chloroform and chloroform and ethanolprecipitation. The purified, radioactively labelled DNA fragments werethen used, with the aid of the transferrin-polylysine transportingsystem, to determine the uptake and inhibition of the erbB-RNA. Thevector pSPT 18 was used as the control DNA.

The test cell system used was a chicken erythroblast cell linetransformed by a temperature-sensitive mutant (ts 34, Graf et al. 1978)of the avine erythroblastosis virus AEV (Beug et al, 1982 b). The erbAoncogene which is also expressed in these cells can be inhibited by aspecific protein kinase inhibitor (H 7). (It was found that the v-erbAoncogene is phosphorylated in vivo and in vitro (i.e. as a bacteriallyexpressed protein) at two sites, namely Ser 28 and Ser29, by proteinkinase C or by cAMP-dependent protein kinase. Mutation of these serinesto form alanines prevents phosphorylation and destroys the v-erbAoncogene activity. H7 is a specific inhibitor of these two kinases andis capable of selectively stopping the changes caused by v-erbA (e.g.blocking of differentiation) in erythroblasts which containv-erbA-v-erbB.)

It is known that erythroblasts in which the erbB oncogene isinactivated--e.g. by increasing the temperature in the case of atemperature-sensitive erbB mutant, are induced to mature intoerythrocytes. One of the first indications of this process is theinduction of haemoglobin synthesis, which can be detected by a sensitivetest (acidic benzidine staining, Orkin et al, 1975, Graf et al, 1978) atthe level of the single cells. Thus, a specific increase in the numberof benzidine-positive cells might be expected, as a phenotypical effectof a ribozyme directed against erbB in this test system.

The test series on which this Example is based was carried out asfollows: the various DNA preparations (see above and Table D), dissolvedin 30 μl of TE buffer, were mixed with 10 μg of native transferrin-ironcomplex or transferrin-polylysine conjugate (dissolved in 50 μl of twicedistilled water) and then incubated for 30 minutes at 37° C.

In the case of the vector DNA (10 μg), correspondingly more (100 μg) ofthe transferrin preparations was used. The DNA transferrin-DNA mixtureswere added to 1 ml of transferrin-free differentiating medium (Zenke etal., 1988). The test cells (per batch 3×10⁶) were incubated before thetest for 60 minutes in transferrin-free differentiating medium at 42° C.(to intensify the uptake of transferrin) and added to theDNA-transferrin-containing mixtures. After 6 hours, 18 hours and 68hours (see below for treatment of cells), samples were taken asdescribed, separated into supernatant and cell sediment, taken up inPK/SDS-buffer and the DNA was analysed.

FIG. 12 shows that, analogously to Example 8, in the cell sample treatedwith transferrin-polylysine, about 5-10 times more DNA was absorbed bythe cells than in the control samples with native transferrin.

Trace m: molecular weight marker: pBR322 DNA, cleaved with HpaII andradioactively labelled using the klenow fragment of DNA polymerase withalpha-³² P-CTP (Maniatis)

Trace 1: 2000 cpm ES13 fragment

Trace 2: material from cells treated with transferrin and ES13

Trace 3: material from cells treated with transferrin-polylysine andES13

After the end of incubation (6 hours) the cells were centrifuged off andincubated in transferrin-containing differentiating medium witherythropoietin and insulin (Kowenz et al, 1986, Zenke et al 1988, 2 mlper batch and at 37° C., i.e. in the presence of an active v-erbBprotein) for a further 72 hours.

The following results were obtained

1. As in Example 8, an increased uptake of DNA could be observed in thesize of the erbB cut DNAs in the cell samples treated withtransferrin-polylysine (about a 5-fold increase).

2. Table D shows that in every case where erbB cut ribozyme tDNA wasintroduced into erbB transformed erythroblasts with the aid ofpolylysine-transferrin constructs, the percentage of benzidine-positivecells was significantly increased (approximately doubled) (the standardused was the samples treated with vector DNA in which the use ofpolylysine-transferrin conjugates, as expected, did not lead to anyincrease in the number of benzidine-positive cells).

EXAMPLE 11

Efficient binding and internalisation of transferrin-polylysine/DNAcomplexes in haematopoietic chicken cells.

The binding of TfpL and TfpL-DNA to cell surface receptors was measuredwith tritium-labelled substances using the method described by Stein etal., 1984. ³ H-labelled TfpL was prepared by conjugation of labelledpolylysine with transferrin using the method described in Example 1. Thelabelling of polylysine 90 was carried out by treating with formaldehydeand ³ H-labelled sodium borohydride (Ascoli and Puet, 1974). The resultsof these tests are shown in FIG. 13. Labelled TfpL 90 (squares) orlabelled TfpL 90 complexed with pB-SK- DNA (Promega Biotech, prepared byTriton-X lysis, CSCl/EtBr equilibrium density gradient centrifugation,decolorisation with 1-butanol and dialysis against 10 mM Tris/HCl pH7.5, 1 mM EDTA) (triangles) were investigated for their specific bindingto the transferrin receptor of HD3 cells. For this purpose theconjugates or complexes (0.1-100 nM) were added to HD3 cells (1×10⁶ /mlin MEM (Eagle's Minimum Medium)+1% BSA) and incubated for 3 hours. FIG.13 shows that both the conjugates and also the complexes bind to HD3cells in such a way that saturation occurs. The apparent bindingconstants calculated from these data amounted to 22 nM for TfpL and 43nM for TfpL-DNA complexes. Although somewhat higher, these valuescorrespond relatively well to those obtained for native transferrin,which were found to be 15 nM.

In order to monitor the uptake of TfpL-DNA complexes into intracellularvesicles, first of all HD3 cells were incubated at 37° C. for 18 hourswith transferrin-free differentiating medium. After the addition of FITCtransferrin or TfpL conjugates (labelled with FITC at the polylysinegroup, and complexed with DNA in some experiments), the cells wereincubated for a further 18 hours. The cells were cytocentrifuged, fixedwith a mixture of 3.7% formaldehyde and 0.02% glutaraldehyde, washedwith PBS, embedded in Mowiol 4.88 and examined under a Zeiss AxiophotFluorescence Microscope. The control used consisted of FITC-labelledgoat anti-mouse antibodies (0.1 mg/ml) (see Example 5). For quantitativedetermination of FITC-Tf, FITC-TfpL and FITC-TfpL/DNA, the cells wereincubated for 6 hours with the preparation in question (Tf: 40 μg/ml;TfpL270: 50 μg/ml; TfpL270f plus pB-SK-DNA (Promega Biotech, prepared byTriton-X lysis, CSCl/EtBr equilibriumn density gradient centrifugation,decolorising with 1-butanol and dialysis against 100 mM Tris/HCl pH 7.5,1 mM EDTA): 50 μg/ml or 16 μg/ml; binding buffer), washed 3 times incold PBS/BSA and subjected to quantitative FACS analysis in aBecton-Dickinson (BD) FACSAN.

FIG. 14 shows that both with TfpL and with TfpL-DNA, all the cells havea relative fluorescence increased more than 10 times, indicating thatthe conjugates or complexes have been taken up by more than 95% of thecell s (Tf: . . . ; TfpL: . . . ; TfpL/DNA:₋₋ ; binding buffer:₋₋).

EXAMPLE 12

Expression of DNA absorbed into the cell by means of TfpL

After it had been established in the preceding Examples that the genetransfer with TfpL is not harmful to cell growth, the activity ofTfpL-DNA complexes which were used themselves for a longer period oftime was tested (the DNA used was plasmid DNA containing the luciferasegene, as described in Example 9). In this test, the same concentrationof HD3 cells was incubated for 1 to 4 days with or without dailysupplementing with TfpL-DNA complexes.

At various intervals of time, aliquots were investigated for lucerifaseenzyme activity, as described in Example 9. In the cultures withrepeated addition of the complexes, a relatively high level ofluciferase gene expression was measured (100,000 to 200,000 light unitsper 10⁷ cells), which remain substantially constant throughout theentire observation period. During this period, no cytotoxic effects wereobserved. If cells were charged with the complexes only once, theluciferase activity decreased between the 2nd and 4th days by a factorof 10 to 20. These results show that in spite of the obviously transientexpression of the luciferase gene, introduced into the cell with the aidof the conjugates according to the invention, a constantly highexpression of the introduced genes can be maintained by repeatedaddition.

EXAMPLE 13

In order to establish how large a proportion of cells actually expressplasmid DNA introduced by transferrin infection, HD3 cells wereincubated with TfpL/DNA complexes as described in the precedingExamples. pRSV-βGal plasmid DNA (₋₋) was used as the DNA. The expressionof this reporter gene was then investigated in individual cells by FACSanalysis (Nolan et al., 1986). TfpL-pB-SK-DNA (. . . ) was used as thecontrol. The fluorescence βGal substrate FDG(fluorescene-Di-β-d-galactopyranoside) was introduced by osmotic shockand the distribution of cells containing fluorescene which was releasedfrom FDG by the βGal enzyme activity was investigated. Uniformdistribution of fluorescene-containing cells leads to the conclusionthat a large proportion of cells express the βGal reporter gene. Theresults of these tests are shown in FIG. 15.

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                                      TABLE A                                     __________________________________________________________________________    Transporting of Polylysin-transferrin into erythroblasts                                      Vesicle     Viability                                                Transferrin-                                                                           fluorescence                                                                              (3H Td R-incorporation)                           Batch                                                                            Medium                                                                            polyLysin                                                                              24 h  48 h  48 h                                              __________________________________________________________________________    1  2 ml                                                                              without addition                                                                       <1%   <1%   140.000 cpm                                       2  2 ml                                                                              145 μl H.sub.2 O                                                                    <1%   <1%   126.000 cpm                                       3  2 ml                                                                              145 μl TfpL Fr1                                                                     >90%++                                                                              >90%++                                                                              137.000 cpm                                       4  2 ml                                                                              145 μl TfpL Fr2                                                                     >90%+++                                                                             >90%+++                                                                             161.000 cpm                                       5  2 ml                                                                              145 μl TfpL Fr3                                                                     >90%+++                                                                             >90%+++                                                                             153.000 cpm                                       6  2 ml                                                                              145 μl TfpL Fr4                                                                     ca. 80%+++                                                                          >90%+++                                                                             151.000 cpm                                       7  2 ml                                                                              145 μl TfpL Fr5                                                                     ca. 60%+++                                                                          >90%+++                                                                             153.000 cpm                                       8  2 ml                                                                              145 μl TfpL Fr6                                                                     ca. 40%+++                                                                          >90%+++                                                                             165.000 cpm                                       __________________________________________________________________________     ++ and +++ indicate the relative intensity of the vesicle fluorescence   

                  TABLE B                                                         ______________________________________                                        PolyLysin-Transferrin can functionally replace normal Transferrin in          the stimulation of the in vitro-induced maturation of erythroblasts                                           Degree of                                                                     maturity                                                    No of cells                                                                           Hemoglobin                                                                              % reticu-                                                   (× 10.sup.6 /ml)                                                                E 492         locytes                                   No   Medium  Addition.sup.b                                                                           24 h 48 h 24 h 48 h 72 h                              ______________________________________                                        1    2 ml    Fe-Transferrin                                                                           3,28 4,38 1,38 2,74 >80%                              2    2 ml    --         2,62 2,56 0,35 0,23  <1%                              3    2 ml    H.sub.2 O  2,60 2,42 0,30 0,13  <1%                              4    2 ml    TfpL Fr1   3,70 4,44 1,36 2,69 >80%                              5    2 ml    TfpL Fr2   3,56 4,24 1,16 2,51 n.b..sup.a                        6    2 ml    TfpL Fr3   3,72 4,54 1,58 2,54 >80%                              7    2 ml    TfpL Fr4   3,48 4,56 1,57 2,55 n.b.                              8    2 ml    TfpL Fr5   3,36 4,26 1,41 2,47 n.b.                              9    2 ml    TfpL Fr6   3,58 4,4  1,14 1,93 60-65%                            ______________________________________                                         .sup.a : not determined                                                       .sup.b : FeTransferrin, 200 μg in 13 μl; TfpL fraction 200 μg in     130 μl; H.sub.2 O, 130 μl                                          

                                      TABLE C                                     __________________________________________________________________________             Medium- Addition    Differentiation parameter                                 Transferrin   Cell number                                                                         Hb  % desint.                                                                          %   %                                   Test                                                                             Transferrin                                                                         Conjugate                                                                              DNA  (× 10.sup.6 /ml)                                                              (E.sup.492)                                                                       Cells                                                                              LR + E                                                                            Ebl                                 __________________________________________________________________________    1  -     --       --   2.56  0.259                                                                             56   <1  44                                     +     --       --   3.72  1.997                                                                             3    73  1                                      -     Tfp190   --   3.67  1.105                                                                             5    54  8                                      -     Tfp1270  --   3.30  1.366                                                                             11   60  4                                   2  -     --       pRSVLuc                                                                            1.24  0.28                                                                              n.n.                                            +     --       pRSVLuc                                                                            5.22  2.459                                                                             n.n.                                            -     Tfp190   pRSVLuc                                                                            4.46  2.265                                                                             n.n.                                         3  -     --       --   2.1   0.222                                                                             79   <1  21                                     +     --       --   2.55  1.369                                                                             6    72  0                                      -     Tfp190   --   2.64  1.016                                                                             10   56  7                                      -     Tf-Prot  --   2.76  1.055                                                                             9    72  4                                   __________________________________________________________________________

                                      TABLE D                                     __________________________________________________________________________    Maturation (hemoglobin content) of v-erbB - transformed erythroblasts         which have absorbed v-erbB - ribozyme DNA                                                                 Hemoglobin content                                                            (% positive of the acetic                         DNA                Transferrin                                                                            benzidine staining                                No                                                                              Type   MW  Amount                                                                              Type Amount                                                                            14 h                                                                              62 h                                          __________________________________________________________________________    1 erb-cut   13                                                                         2 × 10.sup.5                                                                 1 μg                                                                            Tf   10 μg                                                                          <1  15 +- 3.sup.a (3).sup.b                       2 erb-cut   13     TfpL Fr5                                                                           10 μg                                                                          <1  37 +- 4 (2)                                   3 erb-cut   53                                                                         2 × 10.sup.5                                                                 1 μg                                                                            Tf   10 μg                                                                          <1  25 +- 2 (2)                                   4 erb-cut   53     TfpL Fr5                                                                           10 μg                                                                          <1  42 +- 1 (2)                                   5 Vector without                                                                       2 × 10.sup.6                                                                10 μg                                                                            Tf   100 μg                                                                         <1  23 +- 3 (2)                                     ribozyme                                                                    6 Vector without                                                                           10 μg                                                                            TfpL Fr5                                                                           100 μg                                                                         <1  22 +- 2 (2)                                     ribozyme                                                                    7 erb-cut   13                                                                         53 s.o.                                                                           0.5 + 0.5 μg                                                                     Tf   10 μg                                                                          <1  21 +- 2 (2)                                   8 erb-cut   13                                                                         53  0.5 + 0.5 μg                                                                     TfpL Fr5                                                                           10 μg                                                                          <1  38 +- 2 (2)                                   __________________________________________________________________________     .sup.a : more than 200 cells were counted out for each measurement values     + or - standard deviation                                                     .sup.b : No. of independent measurements                                 

We claim:
 1. A transferrin-polycation-nucleic acid complex capable ofbeing absorbed into a cell by transferrin receptor-mediated endocytosis,wherein said complex is soluble under physiological conditions.
 2. Thecomplex of claim 1, wherein said polycation is selected from the groupconsisting of protamine, synthetic homologous or heterologouspolypeptides, histone, and polyethyleneimine.
 3. The complex of claim 1,wherein said polycation is a histone.
 4. The complex of claim 1, whereinsaid polycation is polylysine.
 5. The complex of claim 1, wherein saidpolycation contains between about 20 and 500 positive charges.
 6. Thecomplex of claim 1, wherein the molar ratio of transferrin to polycationis from about 10:1 to about 1:4.
 7. The complex of claim 5, wherein themolar ratio of transferrin to polycation is from about 10:1 to about1:4.
 8. The complex of claim 1, wherein said nucleic acid is capable ofspecifically inhibiting expression of one or more genes.
 9. The complexof claim 1, wherein said nucleic acid is capable of specificallyinhibiting RNA function.
 10. The complex of claim 1, wherein saidnucleic acid is capable of inhibiting a viral nucleic acid.
 11. Thecomplex of claim 1, wherein said nucleic acid is capable of inhibitingone or more oncogenes.
 12. The complex of claim 1, wherein said nucleicacid is capable of inhibiting one or more genes which control cellgrowth or differentiation.
 13. The complex of claim 1, wherein saidnucleic acid is a ribozyme or a gene encoding said ribozyme.
 14. Thecomplex of claim 13, wherein said complex further comprises a carrierRNA or a gene encoding said carrier RNA.
 15. The complex of claim 1,wherein said nucleic acid comprises a ribozyme gene located within acarrier gene, and said carrier gene encodes tRNA.
 16. The complex ofclaim 1, wherein said nucleic acid comprises an antisenseoligonucleotide.
 17. The complex of claim 16, wherein said nucleic acidfurther comprises a carrier nucleic acid.
 18. The complex of claim 16,wherein said nucleic acid comprises a gene encoding a carrier nucleicacid.
 19. A process for introducing nucleic acids into human or animalcells by transferrin receptor-mediated endocytosis comprising:(a)providing a complex which is soluble under physiological conditions,wherein said complex comprises a transferrin-polycation conjugate andone or more nucleic acids, and (b) contacting said complex with saidcells;wherein contacting said complex with said cells occurs in vitroand said nucleic acids are expressed in said cells.
 20. Thetransferrin-polycation-nucleic acid complex as claimed in claim 1,wherein said polycation is not a histone.